CDER评审官指南《色谱分析方法的验证》Center for Drug uation.doc

Center for Drug Evaluationand Research (CDER)Reviewer GuidanceValidation ofChromatographic MethodsNovember 1994CMC 3TABLE OF CONTENTSI. INTRODUCTION目录 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1II. TYPES OF CHROMATOGRAPHY绪言. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2A. 色谱分类 High Performance Liquid Chromatography (HPLC) 高效液相色谱1. Chiral Chromatography手性液相色谱2. Ion-exchange Chromatography离子交换色谱. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33. Ion-pair/Affinity Chromatography离子对/亲和色谱. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34. Normal Phase Chromatography正相色谱. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35. Reversed Phase Chromatography反相色谱. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36. Size Exclusion Chromatography分子排阻色谱. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4B. Gas Chromatography (GC) 气相色谱 . . . . . . . . . . . . . . . . . . . . . . . . . 4C. Thin-Layer Chromatography (TLC) 薄层色谱 . . . . . . . . . . . . . . . . . . .5III. REFERENCE STANDARDS参考标准 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5IV. PARAMETERS FOR VALIDATION OF HPL CHROMATOGRAPHIC METHODS FOR DRUG SUBSTANCE AND DRUG PRODUCT药物及其制剂HPLC方法验证的参数. . . . .7A. Accuracy准确性. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8B. Detection Limit and Quantitation Limit检出限和定量限 . . . . . . . . . . . . . 8C. Linearity线性. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11D. Precision精密度 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131. Repeatability重复性. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13a. Injection Repeatability进样重复性. . . . . . . . . . . . . . . . . . . .13b. Analysis Repeatability分析重复性. . . . . . . . . . . . . . . . . . . .152. Intermediate Precision组间精密度. . . . . . . . . . . . . . . . . . . . . . . .153. Reproducibility重现性. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16E. Range范围. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16F. Recovery回收率. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16G. Robustness耐久性. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16H. Sample Solution Stability供试品溶液的稳定性. . . . . . . . . . . . . . . . . . . . . .17I. Specificity/selectivity专属性/选择性 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17J. System Suitability Specifications and Tests系统适用性规定和试验 . . . . . 211. Capacity factor容量因子 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .222. Precision/Injection repeatability精密度/进样重复性 . . . . . . . . . . . .223. Relative retention相对保留时间. . . . . . . . . . . . . . . . . . . . . . . . . . . . .224. Resolution分离度. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225. Tailing factor拖尾因子. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .236. Theoretical plate number 理论塔板数 . . . . . . . . . . . . . . . . . . . .26K. General Points to Consider要点 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28V. COMMENTS AND CONCLUSIONS注解和结论. . . . . . . . . . . . . . . . . . . . . . . . . .29VI. ACKNOWLEDGEMENTS致谢. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29VII. REFERENCES参考文献. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .291This guidance has been prepared by the Analytical Methods Technical Committee of the Chemistry Manufacturing Controls Coordinating Committee (CMC CC) of the Center for Drug Evaluation and Research at the Food and Drug Administration. Although this guidance does not create or confer any rights for or on any person and does not operate to bind FDA or the industry, it does represent the agencys current thinking on the validation of chromatographic methods. For additional copies of this guidance, contact the Division of Communications Management, HFD-210, CDER, FDA, 5600 Fishers Lane, Rockville, MD 20857 (Phone: 301-594-1012).Send one self-addressed adhesive label to assist the offices in processing your request. An electronic version of this guidance is also available via Internet the World Wide Web (WWW) ( connect to the FDA Home Page at WWW.FDA.GOV/CDER and go to the “Regulatory Guidance” section).REVIEWER GUIDANCE1 VALIDATION OF CHROMATOGRAPHIC METHODSI. INTRODUCTIONThe purpose of this technical review guide is to present the issues to consider when evaluating chromatographic test methods from a regulatory perspective. The document discusses the points to note and weaknesses of chromatography so that CDER reviewers can ensure that the methods performance claims are properly evaluated, and that sufficient information is available for the field chemist to assess the method. Analytical terms, as defined by the International Conference of Harmonization (ICH), 1993, have been incorporated in this guide.本技术指南的目的是给审评人员审评验证色谱方法的, 该文件讨论色谱方法的要点和不足, 以便CDER的审评人员能够保证方法的良好性能，也使化学工作者了解为通过审评应给出的足够的信息。国际协调会议（ICH）1993年定义的分析术语，已在这一指南中运用。Chromatographic methods are commonly used for the quantitative and qualitative analysis of raw materials, drug substances, drug products and compounds in biological fluids. The components monitored include chiral or achiral drug, process impurities,residual solvents, excipients such as preservatives, degradation products, extractables and leachables from container and closure or manufacturing process, pesticide in drug product from plant origin, and metabolites.色谱方法通常用于原料、药物、药物制剂和生物体液中化合物的定性和定量。涉及的成分包括手性的或非手性的药物、过程杂质、残留溶媒、附加剂如防腐剂、分解产物、从容器和密闭包装或制造过程中带入的可提取和可过滤的杂质、植物药中的农药和代谢物等。The objective of a test method is to generate reliable and accurate data regardless of whether it is for acceptance, release, stability or pharmacokinetics study. Data are generated for the qualitative and quantitative testing during development and postapproval of the drug products. The testing includes the acceptance of raw materials,release of the drug substances and products, in-process testing for quality assurance,and establishment of the expiration dating period.试验方法的目的是得到可信赖的和准确的数据，无论是用于验收、出厂、稳定性或药物动力学研究。得到的数据用于药品开发或批准后的定性和定量，试验包括原料的验收、药物和药物制剂的出厂、过程检验(In- process testing)的质量保证和失效期的建立。Validation of a method is the process by which a method is tested by the developer or user for reliability, accuracy and preciseness of its intended purpose. Data thus generated become part of the methods validation package submitted to CDER. 方法的验证是由药品的开发者或使用者来检验其方法是否达到预期的可靠性、准确度和精密度的过程。得到的数据成为方法的验证资料的一部分交给CDER.。Methods validation should not be a one-time situation to fulfil Agency filing requirements, but the methods should be validated and also designed by the developer or user to ensure ruggedness or robustness. Methods should be reproducible when used by other analysts, on other equivalent equipment, on other days or locations, and throughout the life of the drug product. Data that are generated for acceptance,release, stability, or pharmacokinetics will only be trustworthy if the methods used to generate the data are reliable. The process of validation and method design also should be early in the development cycle before important data are generated.Validation should be on-going in the form of re-validation with method changes.方法的验证对于完成机构满足档案要求不是一次性的,开发者和使用者都应验证其方法的耐用度或耐久性（ruggedness or robustness.），其他的分析者、用其它相当的仪器，在其它的日期或地点，在药品生产期限(有效期)全过程，方法都应能够重现。如果产生数据的方法是可靠的，那么所得到的验收、出厂、稳定性或药物动力学的数据就是可信赖的。验证的过程和方法的设计应在开发过程中重要的数据产生之前，如果方法改变了，还应该再验证。II. TYPES OF CHROMATOGRAPHY色谱类型Chromatography is a technique by which the components in a sample, carried by the liquid or gaseous phase, are resolved by sorption-desorption steps on the stationary phase.色谱是一种技术，通过该技术，样品中的组分载入液相或气相中，通过在固定相上由吸附解吸附来完成。A. High Performance Liquid Chromatography (HPLC) 高效液相色谱 (HPLC)HPL chromatographic separation is based on interaction and differential partition of the sample between the mobile liquid phase and the stationary phase. The commonly used chromatographic methods can be roughly divided into the following groups, not necessarily in order of importance:HPLC分离是基于在样品在流动相液体和固定相之间的不同分配。一般地说HPLC大体分为以下几种(未考虑其重要性顺序)1. Chiral手性液相色谱2. Ion-exchange离子交换色谱3. Ion-pair/affinity离子对/亲和色谱4. Normal phase正相色谱5. Reversed phase反相色谱6. Size exclusion分子排阻色谱1. Chiral Chromatography手性液相色谱Separation of the enantiomers can be achieved on chiral stationary phases by formation of diastereomers via derivatizing agents or mobile phase additives on achiral stationary phases. When used as an impurity test method, the sensitivity is enhanced if the enantiomeric impurity elutes before the enantiomeric drug.分离光学异构体可在手性固定相上，用衍生化试剂或在非手性固定相上用流动相添加剂形成非对对映体来实现。用作杂质试验方法时，如果光学异构体杂质在光学异构体药物之前洗脱,要增加灵敏度。2. Ion-exchange Chromatography离子交换色谱Separation is based on the charge-bearing functional groups,anion exchange for sample negative ion (X-), or cation exchange for sample positive ion (X+). Gradient elution by pH is common.分离基于荷电功能团，样品负离子(X - )为阴离子，样品正离子(X + )为阳离子，一般用pH程序洗脱。3. Ion-pair/Affinity Chromatography离子对/亲和色谱Separation is based on a chemical interaction specific to the target species. The more popular reversed phase mode uses a buffer and an added counter-ion of opposite charge to the sample with separation being influenced by pH, ionic strength, temperature,concentration of and type of organic co-solvent(s). Affinity chromatography, common for macromolecules, employs a ligand (biologically active molecule bonded covalently to the solid matrix) which interacts with its homologous antigen (analyte) as a reversible complex that can be eluted by changing buffer conditions.分离基于与目标样品的专一的化学相互作用。更普遍的反相型用缓冲液和加入的对离子(与被分离的样品荷相反电荷)分离。分离受pH、离子强度、温度、浓度和共存的有机溶剂类型的影响。亲和色谱，一般用于大分子,使用配合体（共价结合在固体基质上的生物活性分子)，与其同类的抗原(分析介质)反应，生成可逆转的复合物,通过改变缓冲条件洗脱。4. Normal Phase Chromatography正相色谱Normal phase chromatography is a chromatographic technique that uses organic solvents for the mobile phase and a polar stationary phase. Here, the less polar components elute faster than the more polar components.正相色谱为用有机溶剂为流动相和极性的固定相。此时较小极性的组分比较大极性组分更快地洗脱。5. Reversed Phase Chromatography反相色谱The test method most commonly submitted to CDER is the reversed phase HPLC method. UV detection is the most common detection technique.报给CDER的最通常的实验方法是反相HPLC方法， 最通常用紫外检测器。Reversed phase chromatography, a bonded phase chromatographic technique, uses water as the base solvent. Separation based on solvent strength and selectivity also may be affected by column temperature and pH. In general, the more polar components elute faster than the less polar components.反相色谱,一种键合相的色谱技术，用水作基本溶剂,选择性也受溶剂强度、柱温和pH的影响，一般来说较大极性比较小极性组分洗脱更快。UV detection can be used with all chromatographic techniques.The concern for this type of detector is the loss of sensitivity with lamp aging, and varying sensitivity at the low level depending on design and/or manufacturer. A point to note is that observations on the HPL chromatograms, by UV detection in combination with reversed-phase HPLC, may not be a true indication of the facts for the following reasons:紫外检测器可以用于所有色谱，这类检测器要注意的是灯老化后的灵敏度降低，其灵敏度因(仪器)的设计和/或者制造厂家的不同有小的变异。需要指出,用紫外检检测器和反相HPLC组合得到的色谱图不一定能真实的反映事实，原因是：Compounds much more polar than the compound of interest may be masked (elute together) in the solvent front/void volume.极性比目标化合物大得多的化合物可能被掩盖(在溶剂前沿或死体积时同时洗脱)。Compounds very less polar than the analyte may elute either late during the chromatographic run or are retained in the column.极性比目标化合物小得多的化合物洗脱出来晚,甚至保留在柱上。Compounds with lower UV extinction coefficients or different wavelength maxima may not be detectable at the low level relative to the visibility of the analyte since only one wavelength is normally monitored.紫外吸收系数较低和最大吸收不同的化合物在检测相对较低浓度的目标分析物时不能被检出,因为通常只有一个检测波长。6. Size Exclusion Chromatography排阻色谱Also known as gel permeation or filtration, separation is based on the molecular size or hydrodynamic volume of the components.Molecules that are too large for the pores of the porous packing material on the column elute first, small molecules that enter the pores elute last, and the elution rates of the rest depend on their relative sizes.也叫凝胶渗漉(permeation)或滤过,分离基于化合物分子大小或水动力学(hydrodynamic)容积。比多孔柱填料孔径大得多的分子最先洗脱，小分子进入孔隙洗脱晚，其余的洗脱速率取决于其分子的相对大小。B. Gas Chromatography (GC)Gas chromatography is based on the volatilized sample transported by the carrier gas as the moving phase through the stationary phase of the column where separation takes place by the sorption/desorption process.气相色谱基于挥发性样品由作为流动相的载气运载，通过色谱柱内的固定相时发生吸附和解吸附过程进行分离。Samples for gas chromatographic analysis are normally low molecular weight compounds that are volatile and stable at high temperature. In this respect, residual solvents in drug substances and drug products are suitable for gas chromatographic analysis. Chemical derivatives can also be formed to achieve volatility and thermal stability.通常气相色谱分析的样品是低分子量化合物，这些化合物是易挥发的和高温时稳定的。在这一方面，药物和药物制剂中的残留溶剂适于气相色谱分析。生成化学衍生物可达到易挥发和热稳定的目的。Common detectors are flame ionization (FID) for carbon-containing compounds, electron capture (ECD) for halogenated compounds, flame photometric (FPD) for compounds containing sulphur or phosphorous and nitrogen-phosphorous (NPD) for compounds containing nitrogen or phosphorous. Chiral separation also can be achieved by gas chromatography. Separation by the packed column is rapidly being replaced by the capillary column that provides improved resolution and analysis speed. The location of the analyte on the gas chromatogram is described by retention time (Rt) which is similar to HPLC.常用的检测器是用于含碳化合物的火焰离子化检测器（FID），用于卤代化合物的电子捕获式检测器（ECD），用于含硫和含磷化合物的火焰光度检测器（FPD），以及用于含氮或磷化合物的氮磷检测器（NPD）。气相色谱也能实现手性分离。填充柱迅速被毛细管取代来改进分离度和分析时间，在气相色谱上分析物位置与HPLC一样,用保留时间（Rt）表示。C. Thin-Layer Chromatography (TLC)Thin-layer chromatography is the simplest of the more common chromatographic techniques. Separation is based on migration of the sample spotted on a coated (stationary phase) plate with one edge dipped in a mixture of solvents (mobile phase). The whole system is contained in an enclosed tank.薄层色谱是一种最简便普通的色谱技术，分离基于在一端浸于溶剂混合物（流动相）中的薄层板（固定相）上点的样品移动进行分离，整个系统在密封的缸中进行。Detection techniques include fluorescence, UV and sprays (universal and specific) for compounds that are not naturally colored. The location of the analyte on the TLC plate is described by the Rf value which is the ratio of the migration distance of the compound of interest to the mobile phase front.对于本身没有颜色的化合物，检出技术包括荧光、紫外和喷雾显色剂（通用的和专一的）。 分析物在薄层板上的位置用Rf值来表示，Rf值为化合物的移动距离与溶剂前沿的比值。Of the three techniques, gas, liquid and thin-layer, TLC is the most universal test method as all components are present on the plate and with appropriate detection techniques, all components can be observed. However, it normally is not as accurate or sensitive as HPLC. TLC has a higher analytical variation than HPLC, although one sees the whole picture when appropriate detection schemes are selected.三种方法，气相、液相和薄层中，薄层色谱是最普通的试验方法，因为薄层板上所有的组分都可用适宜的检测技术检出。然而通常不如HPLC那么准确和灵敏。虽然选用适宜的检测技术，TLC法能见到分析的“全图”(whole picture) ，但比HPLC分析变异较大。III. REFERENCE STANDARDS参考标准品（对照品）A reference standard is a highly purified compound that is well haracterized.Chromatographic methods rely heavily on a reference standard to provide accurate data. Therefore the quality and purity of the reference standard is very important. Two types of reference standards, chemical and nuclidic, exist. With the latter, the radiolabel purity should also be considered as well as the chemical purity.参考标准品为经充分鉴定的高纯度化合物，色谱方法更大程度上依赖参考标准品来提供准确的数据。因而参考标准品的质量和纯度是很重要的，有二类参考标准品，化学的和放射性的。后者应考虑放射标记纯度和化学纯度。As described in the Guideline for Submitting Samples and Analytical Data for Methods Validation, the two categories of chemical reference standards are as follows:按照提交方法验证的样品和分析数据，指南中的二类化学参考标准品如下：USP/NF reference standard that does not need characterization, andUSP / NF参考标准品，不需要鉴定。non-compendial standard that should be of the highest purity that can be obtained by reasonable effort and should be thoroughly characterized to assure its identity, strength, quality and purity.非总目录标准品，应用合理方法制备，并经充分鉴定，以保证其鉴别、含量、质量和纯度达到最高。The points to note are: 应该指出Most USP/NF reference standards do not state the purity of the compound.大多数USP / NF参考标准品未标示化合物纯度。The purity correction factor for non-USP reference standards is recommended to be included in the calculation of the test method.对非USP参考标准品，提出纯度的校正数应包括在试验方法的计算中。In addition to structurally-related impurities from the synthesis process,other process impurities like heavy metals, residual solvents, moisture (bound and unbound), pesticides for products of plant origin, and degradation products can also contribute to the lack of purity in the reference standard.提供的参考标准品中没有以下杂质，诸如合成过程的结构相似的杂质和其它的过程杂质，如重金属、残留溶剂、水分（结合的和非结合的）、植物来源制剂中的农药和分解产物等。The drying of the reference standard before use, if stated in the method,will eliminate residual solvent(s), unbound moisture and sometimes bound moisture (depending on the drying conditions). The drying step is always included for hygroscopic compounds. On the other hand, drying can result in the loss of a hydrate or cause degradation in heat-sensitive compounds.如果在方法中规定，用前参考标准品要干燥除去残留溶剂、非结合水分和有时是结合水（取决于干燥条件），对易潮解的化合物总是包括干燥步骤的。但另一方面干燥可能导致结晶水的损失或引起热敏感化合物的降解。Chromatographic test methods use either external or inter